Insights Into the Redox Sensitivity of Chloroflexi Hup-Hydrogenase Derived From Studies in Escherichia coli: Merits and Pitfalls of Heterologous [NiFe]-Hydrogenase Synthesis
Insights Into the Redox Sensitivity of Chloroflexi Hup-Hydrogenase Derived From Studies in Escherichia coli: Merits and Pitfalls of Heterologous [NiFe]-Hydrogenase Synthesis
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DOI：
10.3389/fmicb.2018.02837
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发表时间：
2018-11
影响因子：
5.2
通讯作者：
Nadya Dragomirova;P. Rothe;Stefan Schwoch;S. Hartwig;Constanze Pinske;R. Sawers
中科院分区：
文献类型：
--
作者：
Nadya Dragomirova;P. Rothe;Stefan Schwoch;S. Hartwig;Constanze Pinske;R. Sawers
麦卡提脱氢球菌的高度氧敏感的吸氢(Hup)氢酶是细胞外结合氢氧化和有机卤化物还原的蛋白质呼吸链的一部分。HupXSL蛋白以前被证明是合成的，并在大肠杆菌中具有酶活性。在这里，我们考察了提供活性Hup酶的生长条件，该酶将H2氧化为苯基紫精(BV)还原，并确定了对这一过程重要的宿主因素。在缺乏大肠杆菌三种主要氢酶的遗传背景下，我们可以证明硒半胱氨酸生物合成所必需的基因的额外缺失导致了Hup酶的失活，这表明Hup活性需要甲酸脱氢酶。HUP的活性依赖于甲酸脱氢酶(FDH-H)的存在，这通常与细胞质中放氢甲酸氢解酶(FHL)复合体有关。进一步的分析表明，如果编码铁氧还蛋白样电子转移蛋白HUPX的基因以及相关的FDH-H的HycB小亚基也被缺失，那么异源Hup活性就可以恢复。这些结果表明，催化HupL亚基和电子转移HupS亚基对BV的酶活性是足够的。因此，在没有FDH-H的情况下，HUPX或HycB蛋白的存在似乎会导致HupSL酶的失活。这可能是因为HUPX或HycB帮助电子转移到苯二酚池或其他氧化还原酶复合体，从而使HupSL异源二聚体保持在持续氧化状态，导致其失活。这一建议得到了以下观察的支持，即在有氧或无氧呼吸条件下的生长不会产生活性HupSL。因此，这些研究为理解这种异源合成的氢酶的氧化还原敏感性提供了一个系统。
The highly oxygen-sensitive hydrogen uptake (Hup) hydrogenase from Dehalococcoides mccartyi forms part of a protein-based respiratory chain coupling hydrogen oxidation with organohalide reduction on the outside of the cell. The HupXSL proteins were previously shown to be synthesized and enzymatically active in Escherichia coli. Here we examined the growth conditions that deliver active Hup enzyme that couples H2 oxidation to benzyl viologen (BV) reduction, and identified host factors important for this process. In a genetic background lacking the three main hydrogenases of E. coli we could show that additional deletion of genes necessary for selenocysteine biosynthesis resulted in inactive Hup enzyme, suggesting requirement of a formate dehydrogenase for Hup activity. Hup activity proved to be dependent on the presence of formate dehydrogenase (Fdh-H), which is typically associated with the H2-evolving formate hydrogenlyase (FHL) complex in the cytoplasm. Further analyses revealed that heterologous Hup activity could be recovered if the genes encoding the ferredoxin-like electron-transfer protein HupX, as well as the related HycB small subunit of Fdh-H were also deleted. These findings indicated that the catalytic HupL and electron-transferring HupS subunits were sufficient for enzyme activity with BV. The presence of the HupX or HycB proteins in the absence of Fdh-H therefore appears to cause inactivation of the HupSL enzyme. This is possibly because HupX or HycB aided transfer of electrons to the quinone pool or other oxidoreductase complexes, thus maintaining the HupSL heterodimer in a continuously oxidized state causing its inactivation. This proposal was supported by the observation that growth under either aerobic or anaerobic respiratory conditions did not yield an active HupSL. These studies thus provide a system to understand the redox sensitivity of this heterologously synthesized hydrogenase.